Quantification Of Whole Genome Sequencing DNA Libraries By qPCR

April 05, 2018

University of Birmingham
Biosciences
Research Assistant

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Quality of Results

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Company:

New England Biolabs

Product Name:

NEBNext Library Quant Kit for Illumina

Catalog Number:

E7630S

I have used NEBNext library quant kit from NEB for quantification of whole genome sequencing DNA libraries. After performing qPCR and calibrating with a standard curve generated by kit's standards, my libraries were of 6.8 nM. I have diluted this 6.8 nM library to 4 nM working library to proceed with denaturing and loading into Illumina MiSeq. Overall, this kit performs well and gave me accurate quantification of DNA libraries.

Experimental Design and Results Summary

Application

Next generation sequencing library quantification by qPCR

Starting Material

Pooled whole genomic DNA library (adaptor ligated, index introduced) of 10 nM (based Qubit quantification)

Protocol Overview

The protocol is very simple. Follow the Kit's instructions manual exactly, set up qPCR reaction and thermal cycling. Perform reactions in 20 microliter volume. Do at least three dilutions of DNA library (1 in 1000, 1 in 10,000 and 1 in 100,000) with triplicate for each dilution. Also, set up standards (comes with the kit). Once qPCR has completed, put Ct values of standards and samples in NEB qPCR quantification tool on NEB website. This will give the quantity of DNA library.

Tips

Thaw qPCR reaction mix and standards completely before setting up qPCR reactions.

Results Summary

Quantification of DNA libraries went well with the efficiency of 98% (R^2= 1). Ct values between replicates were spot on and found that my DNA library was of 6.8 nM quantity.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Simple and easy to follow protocol. Calculation of library quantity using online tool is an added advantage.

The Bad

None

The Bottom Line

Whole genomic sequencing DNA libraries were quantified most accurately using NEBNext library quant kit. Based this quantification, I have loaded 20 pM denatured library for Illumina Miseq sequencing and my sequencing went well in optimal cluster density and more than 80 percent of reads having a Q30 score.

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